CN115166230A - Method for improving efficiency of magnetic particle coupled protein by using dendrimer - Google Patents

Method for improving efficiency of magnetic particle coupled protein by using dendrimer Download PDF

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CN115166230A
CN115166230A CN202210778473.9A CN202210778473A CN115166230A CN 115166230 A CN115166230 A CN 115166230A CN 202210778473 A CN202210778473 A CN 202210778473A CN 115166230 A CN115166230 A CN 115166230A
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dendrimer
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李冰
董亚玲
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Zhengzhou Lingsi Biotechnology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

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Abstract

The invention discloses a method for improving the coating efficiency of magnetic particles by using a tree-shaped compound, which comprises the following steps: (1) Mixing the dendrimer with the protein for reaction to obtain a protein-dendrimer conjugate; (2) Mixing the protein-dendrimer conjugate with the magnetic particles for reaction, adsorbing the magnetic particles at the bottom by using a magnet after the reaction is finished, removing supernatant, adding buffer solution for heavy suspension, and then adding sealant for sealing to obtain the protein-dendrimer conjugate. The invention uses the dendrimer as a carrier, so that the labeled protein is connected to the surface of the magnetic particle through chemical bonds. Compared with the traditional magnetic particle coating method, the method can reduce the amount of protein bound to the surface of the magnetic particle by physical adsorption through chemical connection; on the other hand, the dendritic compound has a large number of functional groups on the surface, can be combined with more proteins, has a cascade amplification effect in the magnetic particle coupling process, and greatly improves the sensitivity.

Description

Method for improving efficiency of magnetic particle coupled protein by using dendrimer
Technical Field
The invention relates to a magnetic particle chemiluminescence immunoassay method, in particular to a method for improving the coating efficiency of magnetic particles by using a tree-shaped compound.
Background
At present, the magnetic particle chemiluminescence platform method is widely applied to the fields of in-vitro diagnosis, food safety and the like, and the method mainly takes magnetic particles as solid phase carriers and coupling protein or nucleic acid molecules and the like to detect objects to be detected.
Most of the common magnetic particle surfaces are carboxyl or amino functional groups, and the coupling process with proteins is also a carbodiimide activation method and a glutaraldehyde method, but due to the characteristics of magnetic bead surface modification molecules, more than 30% of protein molecules are still adsorbed on the magnetic particle surfaces through hydrophobic interaction and electrostatic interaction.
The protein-magnetic particle compound adsorbed by physics is unstable in liquid preservation solution, and the protein is easy to fall off from the surface of the magnetic particle, so that the detection signal value is reduced, the stability of the product is influenced in the aspect of the product, and the detection result is inaccurate.
In response to such a situation, many manufacturers often remove non-chemically coupled proteins or nucleic acid molecules by elution after coupling, but accordingly, the coupled proteins on the surface of the magnetic beads are reduced, the detection luminescence signal value is reduced, and the detection sensitivity is also reduced.
There are other methods to modify the surface of the magnetic particles, such as wrapping the surface of the magnetic particles with SiO 2 When the modified graphene is modified, physical adsorption can be reduced; and the surface microspheres are modified by PEG, so that the surface adsorption of the microspheres can be reduced. However, for the in vitro diagnosis manufacturers, the operation process is complex, the in vitro diagnosis manufacturers are more concerned with the application, the in vitro diagnosis manufacturers lack talents for modifying the surface of the magnetic particles, and most manufacturers are reluctant to invest more financial research; on the other hand, the manufacturers for producing the microspheres in batches on the market are few, and the magnetic particles belong to the core raw materials of in vitro diagnosis manufacturers, so that the manufacturers cannot easily change the raw material suppliers.
Therefore, a magnetic particle protein or nucleic acid coupling process which can reduce physical adsorption and does not reduce detection sensitivity is urgently needed to be developed and applied.
Disclosure of Invention
The invention aims to provide a method for improving the coating efficiency of magnetic particles by using a dendrimer, wherein the dendrimer is used as a carrier, and a marker protein is connected to the surface of the magnetic particles through chemical bonds, so that physical adsorption can be reduced, and the detection sensitivity is not reduced.
The invention is realized by the following technical scheme: a method for improving the coating efficiency of magnetic particles by using a dendrimer, comprising the following steps:
(1) Mixing the dendrimer with the protein for reaction to obtain a protein-dendrimer conjugate;
(2) Mixing the protein-dendrimer conjugate with the magnetic particles for reaction, adsorbing the magnetic particles at the bottom by using a magnet after the reaction is finished, removing supernatant, adding buffer solution for heavy suspension, and then adding a sealing agent for sealing to obtain the protein-dendrimer conjugate.
The invention couples antigen or antibody to the surface of magnetic particle by using dendrimer as carrier. Dendrimers are macromolecules with dendritic structures and generally consist of three parts, namely an inner core, a branch unit and a functional group with carboxyl or amino on the surface. By using the dendrimer as a carrier, on one hand, the number of proteins bound to the surface of the magnetic particles by physical adsorption can be reduced through chemical connection; on the other hand, the dendritic compound has a large number of functional groups on the surface, can be combined with more proteins, has a cascade amplification effect in the magnetic particle coupling process, and greatly improves the sensitivity.
The dendrimer compound, namely the dendritic Polyamidoamine (PAMAM), has an amino functional group or a carboxyl functional group on the surface, namely the dendrimer compound is classified into an amino dendrimer compound or a carboxyl dendrimer compound. Wherein, PAMAM with different numbers of iterations is used in the invention, and the amino dendrimer comprises CYD-120A (C) 142 H 288 N 58 O 28 )、CYD-130A(C 302 H 608 N 122 O 60 )、CYD-140A(C 622 H 1248 N 250 O 124 )、CYD-150A(C 1262 H 2528 N 506 O 252 ) The carboxyl dendrimer includes CYD-120C (C) 206 H 352 N 58 O 76 )、CYD-130C(C 430 H 736 N 122 O 156 )、CYD-140C(C 878 H 1504 N 250 O 316 )。
The specific operation of the present invention is different among different types of dendrimers, specifically, the carboxyl dendrimers need to be treated by a cross-linking agent before being coupled with protein; the amino dendrimers can then be directly coupled to proteins.
1. Carboxy dendrimers
(1) Mixing the carboxyl dendrimer solution with a cross-linking agent, carrying out oscillation reaction for 20-40 minutes, and then carrying out mixed reaction with protein to obtain a protein-dendrimer conjugate;
(2) Mixing the protein-dendrimer conjugate with the magnetic particles for reaction, adsorbing the magnetic particles at the bottom by using a magnet after the reaction is finished, removing supernatant, adding buffer solution for heavy suspension, and then adding a sealing agent for sealing to obtain the protein-dendrimer conjugate.
As an example, the carboxyl dendrimer solution is prepared from a carboxyl dendrimer and a MES buffer solution, and the concentration of the carboxyl dendrimer is 1mg/mL; the cross-linking agent is dissolved by adopting a pH 5.0 and 0.1M MES buffer solution until the concentration is 10-70mg/mL, and then is mixed with a carboxyl dendrimer solution, wherein the final concentration of the carboxyl dendrimer is 1-10mg/mL.
The crosslinking agent comprises 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).
In step (1) of the present invention, the final molar ratio of the conjugated protein to the dendrimer is 5.
2. Amino dendrimers
(1) Adding the protein into the amino dendrimer solution, then adding glutaraldehyde, and standing at room temperature for reaction to obtain a protein-dendrimer conjugate;
(2) Mixing the protein-dendrimer conjugate with the magnetic particles treated by the cross-linking agent for reaction, adsorbing the magnetic particles at the bottom by using a magnet after the reaction is finished, removing supernatant, adding buffer solution for heavy suspension, and then adding a sealing agent for sealing to obtain the protein-dendrimer conjugate.
In the step (1), the molar ratio of the glutaraldehyde to the protein to the amino dendrimer is 3-10.
The amino dendrimer solution is prepared from an amino dendrimer and a MES buffer solution, and the concentration of the amino dendrimer is 1mg/mL.
In the step (2), the cross-linking agent is dissolved to the concentration of 10-70mg/mL by using 0.1M MES buffer solution with the pH of 5.0, and then is mixed with the magnetic particles, wherein the final concentration of the magnetic particles is 1-10mg/mL, the reaction is carried out at room temperature by shaking, then the magnetic particles are adsorbed at the bottom by using a magnet, the supernatant is removed, and the MES buffer solution is used for re-suspending the magnetic particles.
The crosslinking agent comprises 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).
The protein of the present invention includes an antibody or an antigen, etc.
The blocking agent is a buffer solution containing blocking protein, and the blocking protein can be BSA, casein, skimmed milk powder and the like.
Compared with the prior art, the invention has the beneficial effects that:
the method is convenient and effective, and the labeled protein is connected to the surface of the magnetic particle through chemical bonds by taking the dendrimer as a carrier. Compared with the traditional magnetic particle coating method, the method can reduce the amount of protein bound to the surface of the magnetic particle by physical adsorption through chemical connection; on the other hand, the dendritic compound has a large number of functional groups on the surface, can be combined with more proteins, has a cascade amplification effect in the magnetic particle coupling process, and greatly improves the sensitivity.
Detailed Description
The present invention will be specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
EXAMPLE A carboxyl surface functional group dendrimer conjugated PCT antibody
1. Dilution of dendrimer: the dendrimer was diluted to 1mg/mL with 0.1M MES (pH 5.5).
2. Treatment of dendrimers: EDC and NHS were prepared in 50mg/mL solution using 0.1M MES (pH 5.5). Then, the diluted dendrimer solution was added to a final concentration of 1 to 10mg/mL, and the reaction was performed for 30 minutes with shaking.
3. Coupling of dendrimers to proteins: and adding a PCT coating antibody into the treated dendritic compound, mixing the PCT coating antibody and the dendritic compound according to the final molar ratio of 10.
4. Cleaning the amino microspheres: putting 200ul of magnetic particles with amino groups on the surface in a glass bottle, adsorbing the magnetic particles at the bottom of the glass bottle by using a magnet, and removing the supernatant; 2ml of 0.02M PBS (pH 8.0) was added to resuspend the magnetic microparticles, and the above procedure was repeated 3 times.
5. Binding of the coupled dendrimer to the magnetic microparticles: and adding the PCT coated antibody-dendrimer conjugate into the washed magnetic particles, and oscillating at room temperature for 2 hours. The amount of PCT-coated antibody-dendrimer conjugate added was calculated from the amount of PCT-coated antibody, and the final amount of PCT-coated antibody was 0.5mg/mL.
6. After the reaction is finished, adsorbing the magnetic particles at the bottom of the glass bottle by using a magnet, and removing the supernatant; 2ml of 0.02M PBS (pH 8.0) was added to resuspend the magnetic microparticles, and the above procedure was repeated 3 times. After washing, 1% BSA and 0.02% sodium azide in PBS buffer (pH 8.0) were added thereto, and the mixture was stored at 2 to 8 ℃.
EXAMPLES diamino surface functional group dendrimers coupling of PCT antibodies
1. Dilution of dendrimer: the dendrimer was diluted to 1mg/mL with 0.02M PBS (pH 8.0) buffer.
2. Conjugation of dendrimers to antigens or antibodies: the PCT coated antibody was added to the diluted dendrimer, and the PCT coated antibody was mixed with the dendrimer at a final molar ratio of 10. Glutaraldehyde is then added to give a final concentration of 0.2%. Standing at room temperature for 2 hours to obtain the PCT coated antibody-dendrimer conjugate.
3. Pretreatment of magnetic particles: putting 200ul of magnetic particles with carboxyl on the surface in a glass bottle, adsorbing the magnetic particles at the bottom of the glass bottle by using a magnet, and removing the supernatant; 2ml of 0.02M PBS (pH 8.0) was added to resuspend the magnetic microparticles, and the above procedure was repeated 3 times. Dissolving EDC and NHS in 0.1M MES (pH 5.0) buffer solution at a concentration of 50mg/ml, respectively, and adding 1ml of each solution into magnetic particles; the reaction was gently shaken at room temperature for 30 minutes. Adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; further 2ml of 0.1M MES (pH 5.0) buffer was added thereto, and the magnetic particles were resuspended, and the above operation was repeated 2 times.
Binding of pct-coated antibody-dendrimer conjugate to treated magnetic microparticles: mixing the PCT coated antibody-dendrimer conjugate with the treated magnetic particles according to a certain proportion (the molar ratio of the PCT coated antibody-dendrimer conjugate to the carboxyl magnetic particles is 2-5: 1), and carrying out shake reaction at room temperature for 2 hours. The amount of PCT coated antibody-dendrimer conjugate added was calculated from the amount of PCT coated antibody, and the final amount of PCT coated antibody was 0.5mg/mL.
5. After the reaction is finished, adsorbing the magnetic particles at the bottom of the glass bottle by using a magnet, and removing the supernatant; 2ml of 0.02M PBS (pH 8.0) was added, and the magnetic microparticles were resuspended, and the above operation was repeated 3 times. After washing, 1% BSA and 0.02% sodium azide in PBS buffer (pH 8.0) were added, and the mixture was stored at 2 to 8 ℃.
Comparative example PCT antibody-coupled magnetic microparticles
1. Washing and activating magnetic particles: putting 200ul of magnetic particles with carboxyl on the surface in a glass bottle, adsorbing the magnetic particles at the bottom of the glass bottle by a magnet, and removing the supernatant; 2ml of 0.02M PBS (pH 8.0) was added thereto, and the above operation was repeated 3 times. Respectively dissolving EDC and NHS in 0.1M MES (pH 5.0) buffer solution at a concentration of 10-70mg/ml, and adding 1ml of each solution into the magnetic particles; lightly shaking for reaction at room temperature for 30-60 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then, 2ml of 0.1M MES (pH 5.0) buffer was added thereto, and the magnetic particles were resuspended, and the above operation was repeated 2 times.
Coupling of pct antibodies: adding PCT antibody into the activated magnetic particles, wherein the amount of the PCT antibody is 0.5mg, and adding 0.1M MES (pH 5.0) buffer solution to a final volume of 1ml; lightly shaking the mixture at room temperature for reaction for 30 minutes; adsorbing the magnetic particles to the bottom with a magnet, removing the supernatant, washing 3 times with 2ml of 0.02M PBS (pH 8.0); 20ml of PBS buffer (pH 8.0) containing 1% BSA and 0.02% sodium azide was added and stored at 2-8 ℃.
In the aspect of immunoassay, magnetic particle components in a magnetic particle chemiluminescence kit (double antibody sandwich method) of a certain foreign manufacturer are replaced, and on-machine assay is carried out by using the Cosmei SMART 500S. The immunoassay sensitivity results are shown in the following table:
TABLE 1 comparison of detection sensitivity of examples and comparative examples
Numbering S0 S1 S2 S3 QB1 QB2 QB3
Concentration of sample 0ng/mL 0.02ng/mL 0.05ng/mL 0.1ng/mL 0.8ng/mL 4.7ng/mL 8.2ng/mL
Example one 0 0.03 0.05 0.09 0.81 4.76 8.28
Example two 0 0.02 0.04 0.11 0.78 4.69 8.31
Comparative example 0 0 0.8 0.13 0.88 4.73 8.16
Table 2 example-37 ℃ thermal stability results
Numbering S0 S1 S2 S3 QB1 QB2 QB3
Concentration of sample 0ng/mL 0.02ng/mL 0.05ng/mL 0.1ng/mL 0.8ng/mL 4.7ng/mL 8.2ng/mL
3 days 0 0.03 0.05 0.10 0.83 4.76 8.22
7 days 0 0.02 0.04 0.11 0.81 4.62 8.19
10 days 0 0.02 0.05 0.12 0.85 4.61 8.21
14 days 0 0.02 0.05 0.09 0.79 4.62 8.16
21 days 0 0.03 0.06 0.11 0.77 4.61 8.11
28 days 0 0.02 0.04 0.08 0.73 4.60 8.02
TABLE 3 example II 37 ℃ thermal stability results
Figure BDA0003722233060000051
Figure BDA0003722233060000061
TABLE 4 comparative example 37 ℃ thermal stability results
Number of S0 S1 S2 S3 QB1 QB2 QB3
Concentration of sample 0ng/mL 0.02ng/mL 0.05ng/mL 0.1ng/mL 0.8ng/mL 4.7ng/mL 8.2ng/mL
3 days 0 0 0.05 0.13 0.85 4.81 8.21
7 days 0 0 0.06 0.10 0.79 4.68 8.19
10 days 0 0 0.05 0.11 0.77 4.66 7.91
14 days 0 0 0.04 0.09 0.72 4.53 7.86
21 days 0 0 0.03 0.08 0.69 4.17 7.11
28 days 0 0 0.02 0.06 0.61 3.85 6.06
As can be seen from the results in Table 1, the dendrimers of the first embodiment and the second embodiment of the invention are used as carriers, so that the labeled protein is connected to the surface of the magnetic particles through chemical bonds, the detection limit value can reach 0.02ng/mL, and the results have small deviation and high accuracy. The detection limit of the comparative example is 0.1ng/mL, when the sample concentration is 0.8ng/mL, larger deviation also occurs, and when the sample concentration is lower than 0.1ng/mL, larger error occurs in the detection result, so that the detection result is inaccurate.
The results in tables 2-4 show that the samples prepared in the first and second examples of the invention have high stability, and the protein does not have obvious shedding after 28 days, and the influence on the detection luminescence signal value is low. Whereas the comparative example showed significant protein shedding after 28 days, and the protein shedding was more pronounced as the sample concentration increased.
In conclusion, the sensitivity and stability of the magnetic particles coupled by the dendrimer serving as the carrier are greatly improved. The method is easy to operate and suitable for large-scale popularization.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims (10)

1. A method for improving the coating efficiency of magnetic particles by using a tree-shaped compound is characterized by comprising the following steps:
(1) Mixing the dendrimer with the protein for reaction to obtain a protein-dendrimer conjugate;
(2) Mixing the protein-dendrimer conjugate with the magnetic particles for reaction, adsorbing the magnetic particles at the bottom by using a magnet after the reaction is finished, removing supernatant, adding buffer solution for heavy suspension, and then adding sealant for sealing to obtain the protein-dendrimer conjugate.
2. The method for increasing the coating efficiency of magnetic particles using the dendrimer according to claim 1, wherein the dendrimer is an amino dendrimer or a carboxyl dendrimer.
3. The method for improving the coating efficiency of magnetic particles by using the dendrimer as claimed in claim 1, wherein the dendrimer is a carboxyl dendrimer, and the method comprises the following steps:
(1) Mixing the carboxyl dendrimer solution with a cross-linking agent, carrying out oscillation reaction for 20-40 minutes, and then carrying out mixed reaction with protein to obtain a protein-dendrimer conjugate;
(2) Mixing the protein-dendrimer conjugate with the magnetic particles for reaction, adsorbing the magnetic particles at the bottom by using a magnet after the reaction is finished, removing supernatant, adding buffer solution for heavy suspension, and then adding sealant for sealing to obtain the protein-dendrimer conjugate.
4. The method for improving the coating efficiency of magnetic particles by using the dendrimer as claimed in claim 3, wherein the carboxyl dendrimer solution is prepared from a carboxyl dendrimer and a MES buffer, and the concentration of the carboxyl dendrimer is 1mg/mL; the cross-linking agent is dissolved by adopting a pH 5.0 and 0.1M MES buffer solution until the concentration is 10-70mg/mL, and then is mixed with a carboxyl dendrimer solution, wherein the final concentration of the carboxyl dendrimer is 1-10mg/mL.
5. The method of claim 4, wherein the cross-linking agent comprises 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.
6. The method for improving the coating efficiency of the magnetic particles by using the dendrimer according to claim 3, wherein the final molar ratio of the protein to the dendrimer is 5.
7. The method of claim 1, wherein the dendrimer is an amino dendrimer and the method comprises the following steps:
(1) Mixing glutaraldehyde and protein, then adding an amino dendrimer solution, and standing at room temperature for reaction to obtain a protein-dendrimer conjugate;
(2) Mixing the protein-dendrimer conjugate with the magnetic particles treated by the cross-linking agent for reaction, adsorbing the magnetic particles at the bottom by using a magnet after the reaction is finished, removing supernatant, adding buffer solution for heavy suspension, and then adding a sealing agent for sealing to obtain the protein-dendrimer conjugate.
8. The method for improving the coating efficiency of the magnetic particles by using the dendrimer according to claim 4, wherein in the step (1), the molar ratio of the glutaraldehyde to the protein to the amino dendrimer is 3-10; the amino dendrimer solution is prepared from an amino dendrimer and a MES buffer solution, and the concentration of the amino dendrimer is 1mg/mL.
9. The method of claim 8, wherein in the step (2), the cross-linking agent is dissolved in MES buffer solution with pH 5.0 and 0.1M to a concentration of 10-70mg/mL, and then mixed with the magnetic particles with a final concentration of 1-10mg/mL, the reaction is shaken at room temperature, and then the magnetic particles are adsorbed on the bottom by a magnet, the supernatant is removed, and the MES buffer solution is used for re-suspending the magnetic particles.
10. The method of claim 4, wherein the cross-linking agent comprises 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.
CN202210778473.9A 2022-06-30 2022-06-30 Method for improving efficiency of magnetic particle coupled protein by using dendrimer Pending CN115166230A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115856286A (en) * 2022-12-30 2023-03-28 中元汇吉生物技术股份有限公司 Preparation method of high-stability magnetic beads

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115856286A (en) * 2022-12-30 2023-03-28 中元汇吉生物技术股份有限公司 Preparation method of high-stability magnetic beads

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